TY - JOUR
AU - Zhou, Jie
AB - Abstract Autophagy, an innate cellular destructive mechanism, plays crucial roles in plant development and responses to stress. Autophagy is known to be stimulated or suppressed by multiple molecular processes, but the role of phytohormone signaling in autophagy is unclear. Here, we demonstrate that the transcripts of autophagy-related genes (ATGs) and the formation of autophagosomes are triggered by enhanced levels of brassinosteroid (BR). Furthermore, the BR-activated transcription factor brassinazole-resistant1 (BZR1), a positive regulator of the BR signaling pathway, is involved in BR-induced autophagy. Treatment with BR enhanced the formation of autophagosomes and the transcripts of ATGs in BZR1-overexpressing plants, while the effects of BR were compromised in BZR1-silenced plants. Yeast one-hybrid analysis and chromatin immunoprecipitation coupled with quantitative polymerase chain reaction revealed that BZR1 bound to the promoters of ATG2 and ATG6. The BR-induced formation of autophagosomes decreased in ATG2- and ATG6-silenced plants. Moreover, exogenous application of BR enhanced chlorophyll content and autophagosome formation and decreased the accumulation of ubiquitinated proteins under nitrogen starvation. Leaf chlorosis and chlorophyll degradation were exacerbated in BZR1-silenced plants and the BR biosynthetic mutant d^im but were alleviated in BZR1- and BZR1-1D-overexpressing plants under nitrogen starvation. Meanwhile, nitrogen starvation-induced expression of ATGs and autophagosome formation were compromised in both BZR1-silenced and d^im plants but were increased in BZR1- and BZR1-1D-overexpressing plants. Taken together, our results suggest that BZR1-dependent BR signaling up-regulates the expression of ATGs and autophagosome formation, which plays a critical role in the plant response to nitrogen starvation in tomato (Solanum lycopersicum). Autophagy is an evolutionarily conserved and highly regulated self-degradation process that recycles cellular nutrients or breaks down damaged components for the optimization of plant growth, development, and stress responses (Qin et al., 2007; Liu and Bassham, 2012; Zhou et al., 2013). In plant cells, autophagy is initiated by the formation of double-membrane vesicles termed autophagosomes, which engulf the intracellular material and subsequently deliver them to vacuoles for degradation under stress, such as nutrient starvation (Bassham et al., 2006; Hofius et al., 2009; Araújo et al., 2011). The deficiency of the autophagic genes is associated with susceptibility to nitrogen (N) and carbon starvation and suppressed senescence-induced breakdown of mitochondria-resident proteins in plants (Zhou et al., 2013; Li et al., 2014). Autophagy-deficient mutants had increased the levels of insoluble proteins that are highly ubiquitinated under heat and oxidative stresses in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum; Zhou et al., 2014b). Furthermore, autophagy has been shown to interact with defense signaling pathways and induce plant resistance against pathogens (Liu et al., 2005; Lai et al., 2011). In recent years, the identification of autophagy-related genes (ATGs) has firmly established the occurrence of autophagosome formation. However, our understanding of the mechanisms and signaling cascades that regulate autophagy in plants remains incomplete (Thompson and Vierstra, 2005; Michaeli et al., 2016). Target of rapamycin (TOR), a PtdIns3K-related kinase that can phosphorylate Atg13 and inhibit the formation of the Atg1/13 complex, has been identified as a key regulator of autophagy in plants (Liu and Bassham, 2010, 2012; Pérez-Pérez et al., 2010). TOR RNA interference Arabidopsis plants showed constitutive activation of autophagy (Liu and Bassham, 2010). NBR1 (a neighbor of the BRCA1 gene), the first identified cargo receptor for selective autophagy, interacts with both Atg8 and ubiquitin and mediates the encapsulation of ubiquitinated protein aggregates in autophagosomes (Svenning et al., 2011; Zientara-Rytter et al., 2011; Zhou et al., 2013). Arabidopsis nbr1 mutants are selectively hypersensitive to specific abiotic stresses, including heat, oxidative stress, and osmotic stress, but no difference is observed between nbr1 and wild-type plants in response to age- and darkness-induced senescence or necrotrophic pathogens (Zhou et al., 2013). In addition, heat shock transcription factor A1a was reported to bind to the promoters of ATGs and induce their expression, resulting in autophagosome formation and, eventually, increased drought tolerance in tomato (Wang et al., 2015). Notably, reactive oxygen species also are involved in the induction of autophagy in plants (Xiong et al., 2007; Chen et al., 2015). Glyceraldehyde-3-phosphate dehydrogenase, an important enzyme in the glycolytic pathway, is thought to transduce hydrogen peroxide signal and can antagonistically regulate autophagy in plants (Guo et al., 2012; Henry et al., 2015). The cytoplastic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCs) interact with Atg3 to inhibit its activity in Nicotiana benthamiana plants. Meanwhile, reactive oxygen species weaken the interaction between GAPCs and Atg3 but enhance the Atg3-Atg8 interaction and autophagic responses (Han et al., 2015). In animal development and disease resistance, fine regulation of autophagy relies on different hormone signals (Sinha et al., 2012; Tian et al., 2013; Chen et al., 2014). However, the regulation of autophagy in plants by phytohormones and the underlying mechanisms are largely unknown. In Arabidopsis, drought tolerance is induced through ring finger E3 ligase-mediated ubiquitination downstream of stress-responsive abscisic acid signaling (Zhang et al., 2007). Autophagosomes can engulf ubiquitinated proteins and then transfer them to vacuoles for degradation by hydrolytic enzymes. Ethylene treatment increased the expression of ATG8 homologs in petals of petunia (Petunia hybrida), while pollination induced the formation of autophagosomes accompanied by increasing ethylene production (Shibuya et al., 2013). In addition, autophagy was induced by an agonist of salicylic acid, benzo-(1,2,3)-thiadiazole-7-carbothioic acid (Yoshimoto et al., 2009). Taken together, these observations suggest that phytohormones may be involved in the activation of autophagy. However, the mechanism of autophagic induction by phytohormone signaling remains unclear. Brassinosteroids (BRs) are phytohormones that play critical roles in plant growth, development, and responses to stress (Kim and Wang, 2010; Sun et al., 2010; Albrecht et al., 2012). BRs are first perceived by the receptor brassinosteroid-insensitive1 (BRI1); this is followed by autophosphorylation and activation of the BRI1 intracellular kinase domain (Kinoshita et al., 2005; Wang et al., 2014). This activated BRI1 triggers a downstream phosphorylation and dephosphorylation signal transduction cascade that results in the nuclear localization of dephosphorylated brassinazole-resistant1 (BZR1) and BRI1-EMS-suppressor1 transcription factors, which bind to the E-boxes (CANNTG) and/or to the BR-response element (CGTGT/CG) of the promoters of target genes (He et al., 2005; Kim and Wang, 2010; Sun et al., 2010; Jiang et al., 2015). A recent study demonstrated that TOR signaling mediates autophagy to degrade BZR1, which is involved in the regulation of the BR signaling pathway (Zhang et al., 2016). Although BR is a type of multifunctional hormone, its roles in regulating autophagic degradation are unclear. Genome-wide microarray experiments indicated that the expression of approximately 20% of genes is regulated by BR in Arabidopsis (Guo et al., 2013). A number of studies also have demonstrated that BRs activate multiple signaling pathways to induce plant tolerance against various environmental stresses that have similar roles in autophagy during plant stress responses (Choudhary et al., 2012; Lozano-Durán et al., 2013; Zhou et al., 2014a). However, functional evidence regarding the involvement of BR signaling in autophagy pathways is absent. Autophagy plays a vital role in N starvation in plants. The atg mutants, such as atg4, atg5, and atg7, are more sensitive to nutrient-limited conditions than wild-type plants (Yoshimoto et al., 2004; Phillips et al., 2008). atg10-1 plants show dysfunctional accumulation of autophagic bodies in vacuoles under nutrient-deficient conditions and increased sensitivity to N starvation, leading to elevated leaf senescence and programmed cell death (Phillips et al., 2008). Furthermore, Atg11 interacts with the Atg1/13 protein kinase complex to promote the starvation-induced phosphorylation of Atg1 and the turnover of Atg1 and Atg13, which provides a dynamic mechanism that tightly connects autophagy to the nutritional status of plants (Li et al., 2014). The atg12 mutants show altered autophagic transport and N remobilization, leading to the inhibition of seedling growth and plant maturation, increased leaf senescence, and arrested ear development under N starvation in maize (Zea mays; Li et al., 2015). In this study, we found that BR induced the BZR1-mediated formation of autophagy in tomato. Silencing of BZR1 attenuated the transcript levels of ATGs and the formation of autophagosomes, while these parameters were enhanced in BZR1-overexpressing plants after BR treatment. Silencing of ATG2 and ATG6 compromised the formation of BR-induced autophagosomes. In addition, silencing of BZR1 compromised the resistance to N starvation, but this characteristic was enhanced in BZR1-overexpressing and exogenous BR-treated plants. This report demonstrates that the BR signaling pathway positively regulates autophagy in tomato plants through BZR1 activation, while BR signal-induced autophagy plays a vital role in response to N starvation. RESULTS BR Induces Autophagy and ATG Expression To investigate whether BR can induce autophagy in tomato plants, we first analyzed the transcript levels of 12 tomato ATGs after treatment with exogenous brassinolide (BL; the most biologically active member of the BR family). As shown in Figure 1A, the transcript levels of ATGs increased slightly as early as 3 h and reached the highest levels at 12 h after BL treatment. However, the expression levels of most of the ATGs decreased to control levels after BL application for 24 h (Fig. 1A). To gain further insight into the role of BL in activating autophagy, we used the fluorescent dye monodansylcadaverine (MDC) to detect autophagic activity in wild-type plants after BL treatment. In the control plants, only a few MDC-stained autophagosomes were observed (Fig. 1, B and C). In contrast, numerous MDC-stained autophagosomes were detected after BL treatment (Fig. 1, B and C). Then, we used transmission electron microscopy (TEM), which is a classic method for detecting autophagy in most organisms, including plants, to confirm the MDC results. Consistent with the results of MDC staining, TEM showed only a few classic autophagosomes with double membranes in the cytoplasm and single-membrane autophagic bodies in the vacuoles of the control plants (Fig. 1, D and E). Nonetheless, the numbers of autophagosomes and autophagic bodies increased by 9.3-fold at 12 h after BL treatment (Fig. 1, D and E). Notably, during autophagosome formation, the C terminus of Atg8 is cleaved by Atg4 and conjugated to the membrane lipid phosphatidylethanolamine (PE), which is monitored as a marker for autophagic activation (Yoshimoto et al., 2004; Kwon et al., 2013). To verify our results, we analyzed the formation of Atg8-PE conjugates by immunoblotting. Significantly, the Atg8-PE bands were detected in the plants at 12 h after BL treatment, but they were barely found in the control plants (Fig. 1F). Figure 1. Open in new tabDownload slide Effects of BRs on the induction of autophagy in tomato leaves. A, Heat map showing the expression profiles of ATGs after BL treatment at different time points. Six-week-old tomato wild-type cv Condine Red plants were treated with 500 nm BL, and total RNA was extracted from leaf samples at the indicated times. Transcript levels were determined using real-time quantitative PCR (RT-qPCR), and cluster analysis was performed using MeV version 4.9. The color bar at the top shows the levels of expression; 3 h, 6 h, 12 h, and 24 h indicate the time course: 3, 6, 12, and 24 h, respectively, after BL treatment. B, MDC-stained autophagosomes in the leaves of wild-type plants. Six-week-old plants were treated with 500 nm BL, and the leaves were stained with MDC and visualized at 12 h by confocal microscopy. MDC-stained autophagosomes are in green. Bars = 20 μm. C, Relative autophagic activity normalized to the activity of the wild-type control plants in B. The number of MDC-stained autophagosomes per image was quantified to calculate the autophagic activity relative to wild-type control plants, which was set to 1. More than 20 images for each treatment were used for the quantification. D, Representative TEM images of autophagic structures in the mesophyll cells of wild-type plants. Six-week-old plants were treated with 500 nm BL, and the mesophyll cells were visualized at 12 h by TEM. Autophagic bodies are marked by red arrows. S, Starch; V, vacuole. Bars = 1 µm. E, Relative autophagic activity normalized to the activity of the wild-type control plants in D. The number of autophagic bodies per image was quantified to calculate the autophagic activity relative to wild-type control plants, which was set to 1. More than 20 images were used to quantify autophagic structures. F, Atg8 protein levels in the leaves of wild-type plants. The nonlipidated and lipidated forms of Atg8 are indicated by Atg8 and Atg8-PE, respectively. Actin was used as a loading control for the western-blot analysis. The results in C and E represent means ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed with similar results. Figure 1. Open in new tabDownload slide Effects of BRs on the induction of autophagy in tomato leaves. A, Heat map showing the expression profiles of ATGs after BL treatment at different time points. Six-week-old tomato wild-type cv Condine Red plants were treated with 500 nm BL, and total RNA was extracted from leaf samples at the indicated times. Transcript levels were determined using real-time quantitative PCR (RT-qPCR), and cluster analysis was performed using MeV version 4.9. The color bar at the top shows the levels of expression; 3 h, 6 h, 12 h, and 24 h indicate the time course: 3, 6, 12, and 24 h, respectively, after BL treatment. B, MDC-stained autophagosomes in the leaves of wild-type plants. Six-week-old plants were treated with 500 nm BL, and the leaves were stained with MDC and visualized at 12 h by confocal microscopy. MDC-stained autophagosomes are in green. Bars = 20 μm. C, Relative autophagic activity normalized to the activity of the wild-type control plants in B. The number of MDC-stained autophagosomes per image was quantified to calculate the autophagic activity relative to wild-type control plants, which was set to 1. More than 20 images for each treatment were used for the quantification. D, Representative TEM images of autophagic structures in the mesophyll cells of wild-type plants. Six-week-old plants were treated with 500 nm BL, and the mesophyll cells were visualized at 12 h by TEM. Autophagic bodies are marked by red arrows. S, Starch; V, vacuole. Bars = 1 µm. E, Relative autophagic activity normalized to the activity of the wild-type control plants in D. The number of autophagic bodies per image was quantified to calculate the autophagic activity relative to wild-type control plants, which was set to 1. More than 20 images were used to quantify autophagic structures. F, Atg8 protein levels in the leaves of wild-type plants. The nonlipidated and lipidated forms of Atg8 are indicated by Atg8 and Atg8-PE, respectively. Actin was used as a loading control for the western-blot analysis. The results in C and E represent means ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed with similar results. To test the role of endogenous BR in the induction of autophagy, we compared the autophagic activity in the wild type, d^im, a weak allele mutant impaired in the key BR biosynthetic gene DWARF (DWF), and DWF-homozygous T2 progeny of DWF-overexpressing (DWFOE) plants from two independent lines (2# and 3#) with accumulated high levels of endogenous BL (Li et al., 2016). We detected low-punctate fluorescent signals in wild-type and d^im plants; however, the number of MDC-stained autophagosomes increased significantly in DWFOE-2# and DWFOE-3# plants (Supplemental Fig. S1, A and B). The Atg8-PE bands were weak in wild-type and d^im plants but were prominent in DWFOE-2# and DWFOE-3# plants (Supplemental Fig. S1C). Taken together, these results suggest that higher levels of BR, either through exogenous application or endogenous manipulation, can induce the formation of autophagosomes in tomato plants. BZR1 Modulates BR-Induced Autophagy and ATG Expression The BZR1 transcription factor is a downstream component of the BR signal transduction cascade, which regulates thousands of nuclear genes (Belkhadir and Jaillais, 2015). To investigate the role of BZR1 in the BR-induced formation of autophagosomes, we compared BZR1-silenced (TRV-BZR1) plants, which had approximately 20% of the BZR1 transcript level of the TRV control plants (Supplemental Fig. S2A), wild-type plants, and homozygous T2 progeny of BZR1-overexpressing (BZR1OE) plants from two independent lines (1# and 2#). The expression levels of BZR1 in BZR1OE lines (1# and 2#) were 33.8 and 29.8 times higher than those in wild-type plants, respectively (Supplemental Fig. S2B). BZR1 protein was noticeably phosphorylated in BZR1OE plants without BL treatment, while the dephosphorylated bands were increased after BL treatment, especially at 12 h (Supplemental Fig. S3A). Furthermore, the expression levels of CPD and DWF, two key genes involved in BR biosynthesis in BZR1OE plants, were 15.2% to 18.1% lower than those in wild-type plants in the absence of BL, respectively (Supplemental Fig. S3, B and C). The expression levels of CPD and DWF in wild-type plants were decreased by 48% and 53.6%, respectively, after BL treatment for 12 h, but their expression levels were much higher than those in the BZR1OE plants (Supplemental Fig. S3, B and C). Strikingly, we observed few MDC-stained autophagosomes in plants grown in the absence of BL (Fig. 2, A and B). However, silencing of BZR1 significantly suppressed the formation of autophagosomes by BL treatment, as evidenced by staining results at 12 h (Fig. 2, A and B). BZR1OE plants had more MDC-stained autophagosomes than wild-type plants after BL treatment (Fig. 2, A and B). The TEM results were consistent with the MDC staining results, as few autophagosomes and autophagic bodies were observed in all the plants in the absence of BL (Supplemental Fig. S4, A and B). However, the numbers of autophagosomes and autophagic bodies increased from 9.5- to 10-fold in TRV and wild-type plants after 12 h of BL application (Supplemental Fig. S4, A and B). Meanwhile, increases of 2.9- and 18-fold were observed in the TRV-BZR1 plants and BZR1OE plants, respectively (Supplemental Fig. S4, A and B). To further confirm our results, we used western blotting to detect the abundance of Atg8-PE. In control plants, Atg8-PE bands were barely detected (Fig. 2, C and D), while Atg8-PE bands were abundant in BL-treated TRV and wild-type plants (Fig. 2, C and D). Interestingly, the Atg8-PE band was weak in TRV-BZR1 plants but was more prominent in BZR1OE plants at 12 h after BL treatment (Fig. 2, C and D). These results suggest the potential involvement of BZR1 in BR-induced autophagy. Figure 2. Open in new tabDownload slide Accumulation of autophagosomes in BZR1-silenced and BZR1OE plants after BL treatment. A, MDC-stained autophagosomes in the leaves of TRV, TRV-BZR1, wild-type (WT), and BZR1OE plants. Six-week-old plants were treated with 500 nm BL. After 12 h, the leaves were stained with MDC and visualized by confocal microscopy. MDC-stained autophagosomes are shown in green. Bars = 20 μm. B, Relative autophagic activity normalized to the activity of the TRV or wild-type control plants in A. The number of MDC-stained autophagosomes per image was quantified to calculate the autophagic activity relative to TRV or wild-type control plants, which was set to 1. More than 20 images for each treatment were used for the quantification. C and D, Atg8 protein levels in the leaves of TRV and TRV-BZR1 or wild-type and BZR1OE plants. The nonlipidated and lipidated forms of Atg8 are indicated by Atg8 and Atg8-PE, respectively. Actin was used as a loading control for the western-blot analysis. The results in B represent means ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. 1# and 2# represent two lines of BZR1OE plants. Figure 2. Open in new tabDownload slide Accumulation of autophagosomes in BZR1-silenced and BZR1OE plants after BL treatment. A, MDC-stained autophagosomes in the leaves of TRV, TRV-BZR1, wild-type (WT), and BZR1OE plants. Six-week-old plants were treated with 500 nm BL. After 12 h, the leaves were stained with MDC and visualized by confocal microscopy. MDC-stained autophagosomes are shown in green. Bars = 20 μm. B, Relative autophagic activity normalized to the activity of the TRV or wild-type control plants in A. The number of MDC-stained autophagosomes per image was quantified to calculate the autophagic activity relative to TRV or wild-type control plants, which was set to 1. More than 20 images for each treatment were used for the quantification. C and D, Atg8 protein levels in the leaves of TRV and TRV-BZR1 or wild-type and BZR1OE plants. The nonlipidated and lipidated forms of Atg8 are indicated by Atg8 and Atg8-PE, respectively. Actin was used as a loading control for the western-blot analysis. The results in B represent means ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. 1# and 2# represent two lines of BZR1OE plants. To further investigate the role of BZR1 in BR-induced autophagy, we examined the transcript levels of six ATGs in TRV-BZR1 and BZR1OE plants. In the absence of BL, the expression levels of these ATGs in TRV-BZR1 or BZR1OE plants were not significantly different from those in TRV or wild-type plants (Fig. 3). However, the transcripts of ATG2 and ATG6 were induced by 1.5-fold in TRV plants after BL application but decreased by 28.1% and 26.2% in TRV-BZR1 plants compared with TRV plants, respectively, after BL treatment (Fig. 3). In comparison, the expression levels of ATG5, ATG8h, ATG9, and ATG18f were not different from those in TRV plants (Fig. 3A). Strikingly, BL treatment resulted in a more significant increase in the expression of ATGs in BZR1OE plants (Fig. 3B). Taken together, these results suggest that BR regulates autophagy by modulating BZR1-mediated BR signaling and the expression of ATGs. Figure 3. Open in new tabDownload slide Induction of ATGs by BL in BZR1-silenced and BZR1OE plants. A, Expression of ATGs in TRV and TRV-BZR1 plants. B, Expression of ATGs in wild-type (WT) and BZR1OE plants. Six-week-old tomato plants were treated with 500 nm BL, and total RNA was extracted from leaf samples harvested after 12 h. The expression levels were determined using RT-qPCR. All data are presented as means of five biological replicates ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. 1# and 2# represent two lines of BZR1OE plants. Figure 3. Open in new tabDownload slide Induction of ATGs by BL in BZR1-silenced and BZR1OE plants. A, Expression of ATGs in TRV and TRV-BZR1 plants. B, Expression of ATGs in wild-type (WT) and BZR1OE plants. Six-week-old tomato plants were treated with 500 nm BL, and total RNA was extracted from leaf samples harvested after 12 h. The expression levels were determined using RT-qPCR. All data are presented as means of five biological replicates ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. 1# and 2# represent two lines of BZR1OE plants. To further validate the possible regulation of ATGs by BZR1, we examined the promoters of ATG2 and ATG6 and found that their promoters contain E-boxes (CANNTG; Fig. 4A). We performed a yeast one-hybrid assay to determine whether BZR1 can bind directly to the ATG2 and ATG6 promoters in vitro. As shown in Figure 4B, yeast cells containing only the bait vector harboring ATG2 and ATG6 promoter regions grew on selection medium when transformed with BZR1-AD, while those transformed with empty pGADT7 vector did not grow on the selection medium. Meanwhile, yeast cells containing the bait vector harboring mutated ATG2 and ATG6 promoter regions did not grow on selection medium when transformed with BZR1-AD and pGADT7 vector (Supplemental Fig. S5). These results indicate that BZR1 binds directly to the promoters of ATG2 and ATG6 in vitro. To determine whether tomato BZR1 directly regulates the expression of ATG2 and ATG6 in vivo, we used chromatin immunoprecipitation (ChIP) coupled with qPCR assays to analyze BZR1 protein binding to the promoters of both genes with or without BL treatment. Strikingly, only the promoter sequences of ATG2 and ATG6 were precipitated from the chromatin of 3-hemagglutinin (HA)-tagged BZR1OE plants with an anti-HA antibody after BL treatment, but this was not observed in BZR1OE plants without BL treatment and wild-type plants (Fig. 4C). Furthermore, IgG control antibody failed to precipitate these gene promoter sequences (Fig. 4C). Thus, BZR1 binds directly to the ATG2 and ATG6 promoters and may regulate their expression in response to BR stimuli. Figure 4. Open in new tabDownload slide BZR1 binds to the promoters of ATGs in vitro and in vivo. A, E-boxes in the promoters of tomato ATG2 and ATG6. Numbering is from predicted transcriptional start sites. B, Yeast one-hybrid assay showing the binding of BZR1-AD to ATG2 and ATG6 promoters. Yeast cells with positive DNA-protein interactions were grown on Leu− plates with 100 ng mL−1 aureobasidin A. C, Direct binding of BZR1 to the promoters of ATG2 and ATG6 was investigated using ChIP-qPCR in BZR1OE plants. Six-week-old BZR1OE plants were treated with water or 500 nm BL, and input chromatin was isolated from leaf samples at 12 h. An anti-HA antibody was used to immunoprecipitate the epitope-tagged BZR1-chromatin complex, while the control reaction was performed in parallel with mouse IgG. Input and ChIP-DNA samples were analyzed by qPCR using primers specific to the promoters of the ATGs. The ChIP results are presented as percentages of the input DNA. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. WT, Wild type. 1# represents a line of BZR1OE plant. Figure 4. Open in new tabDownload slide BZR1 binds to the promoters of ATGs in vitro and in vivo. A, E-boxes in the promoters of tomato ATG2 and ATG6. Numbering is from predicted transcriptional start sites. B, Yeast one-hybrid assay showing the binding of BZR1-AD to ATG2 and ATG6 promoters. Yeast cells with positive DNA-protein interactions were grown on Leu− plates with 100 ng mL−1 aureobasidin A. C, Direct binding of BZR1 to the promoters of ATG2 and ATG6 was investigated using ChIP-qPCR in BZR1OE plants. Six-week-old BZR1OE plants were treated with water or 500 nm BL, and input chromatin was isolated from leaf samples at 12 h. An anti-HA antibody was used to immunoprecipitate the epitope-tagged BZR1-chromatin complex, while the control reaction was performed in parallel with mouse IgG. Input and ChIP-DNA samples were analyzed by qPCR using primers specific to the promoters of the ATGs. The ChIP results are presented as percentages of the input DNA. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. WT, Wild type. 1# represents a line of BZR1OE plant. BR-Dependent ATGs Are Involved in the Formation of Autophagosomes To further investigate the role of the ATGs in BR-induced autophagy, we silenced ATG2 and ATG6 in wild-type and BZR1OE plants, respectively, and the expression of these genes decreased by 60% to 85% in gene-silenced plants compared with the expression levels in the TRV control plants (Supplemental Fig. S6). BL treatment increased the number of MDC-stained autophagosomes by 8.2-fold in wild-type plants and by 15.8-fold in BZR1OE plants, respectively (Fig. 5, A and B). Furthermore, silencing of ATG2 and ATG6 compromised the BL-induced accumulation of MDC-stained autophagosomes (Fig. 5, A and B). Moreover, BL treatment increased the numbers of autophagosomes and autophagic bodies by 7.2- and 15.8-fold in wild-type and BZR1OE plants, respectively (Fig. 5, C and D). Importantly, BL failed to induce the formation of autophagosomes and autophagic bodies in ATG2- and ATG6-silenced plants (Fig. 5, C and D). Furthermore, the abundance of Atg8-PE was not significantly different in any of the plants in the absence of BL (Fig. 5E). While BL application increased the abundance of Atg8-PE in wild-type and BZR1OE plants, this increase was compromised in both ATG2- and ATG6-silenced plants (Fig. 5E). These results suggest that BL-induced autophagosomes are largely dependent on ATG2 and ATG6. Figure 5. Open in new tabDownload slide Effects of BL on the autophagosome formation in ATG2- and ATG6-silenced plants. A, MDC-stained autophagosomes in the leaves of wild-type (WT) or BZR1OE TRV, TRV-ATG2, and TRV-ATG6 plants. Six-week-old plants were treated with 500 nm BL; after 12 h, the leaves were stained with MDC and visualized by confocal microscopy. MDC-stained autophagosomes are shown in green. Bars = 20 μm. B, Relative autophagic activity normalized to the activity of the wild-type TRV control plants in A. The number of MDC-stained autophagosomes per image was quantified to calculate the autophagic activity relative to wild-type TRV control plants, which was set to 1. More than 20 images for each treatment were used for the quantification. C, TEM images of autophagic structures in the mesophyll cells of wild-type TRV, TRV-ATG2, and TRV-ATG6 or BZR1OE TRV, TRV-ATG2, and TRV-ATG6 plants. Six-week-old plants were treated with 500 nm BL, and the mesophyll cells were visualized after 12 h by TEM. Autophagic bodies are indicated by red arrows. Cp, Chloroplast; S, starch; V, vacuole. Bars = 1 µm. D, Relative autophagic activity normalized to the activity of the wild-type TRV control plants in C. The number of autophagic bodies per image was quantified to calculate the autophagic activity relative to wild-type TRV control plants, which was set to 1. More than 20 images were used to quantify autophagic structures. E, Atg8 protein levels in the leaves of wild-type or BZR1OE TRV, TRV-ATG2, and TRV-ATG6 plants. The nonlipidated and lipidated forms of Atg8 are indicated by Atg8 and Atg8-PE, respectively. Actin was used as a loading control for the western-blot analysis. The results in B and D represent means ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. 1# represents a line of BZR1OE plant. Figure 5. Open in new tabDownload slide Effects of BL on the autophagosome formation in ATG2- and ATG6-silenced plants. A, MDC-stained autophagosomes in the leaves of wild-type (WT) or BZR1OE TRV, TRV-ATG2, and TRV-ATG6 plants. Six-week-old plants were treated with 500 nm BL; after 12 h, the leaves were stained with MDC and visualized by confocal microscopy. MDC-stained autophagosomes are shown in green. Bars = 20 μm. B, Relative autophagic activity normalized to the activity of the wild-type TRV control plants in A. The number of MDC-stained autophagosomes per image was quantified to calculate the autophagic activity relative to wild-type TRV control plants, which was set to 1. More than 20 images for each treatment were used for the quantification. C, TEM images of autophagic structures in the mesophyll cells of wild-type TRV, TRV-ATG2, and TRV-ATG6 or BZR1OE TRV, TRV-ATG2, and TRV-ATG6 plants. Six-week-old plants were treated with 500 nm BL, and the mesophyll cells were visualized after 12 h by TEM. Autophagic bodies are indicated by red arrows. Cp, Chloroplast; S, starch; V, vacuole. Bars = 1 µm. D, Relative autophagic activity normalized to the activity of the wild-type TRV control plants in C. The number of autophagic bodies per image was quantified to calculate the autophagic activity relative to wild-type TRV control plants, which was set to 1. More than 20 images were used to quantify autophagic structures. E, Atg8 protein levels in the leaves of wild-type or BZR1OE TRV, TRV-ATG2, and TRV-ATG6 plants. The nonlipidated and lipidated forms of Atg8 are indicated by Atg8 and Atg8-PE, respectively. Actin was used as a loading control for the western-blot analysis. The results in B and D represent means ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. 1# represents a line of BZR1OE plant. BR-Induced Autophagy Is Essential in N Starvation Autophagy plays vital roles in nutrient recycling, which involves the engulfment of damaged and unfolded proteins or cytoplasmic organelles and their transfer to vacuoles for reuse (Liu and Bassham, 2012). To better understand the role of BR-induced autophagy, we tested plant tolerance to N starvation after BR treatment. As shown in Figure 6A, no significant difference was observed under optimal growth conditions, while N starvation dramatically attenuated plant growth, with leaves showing chlorosis. The chlorophyll content in wild-type plants was decreased by 52.2% at day 14 after N starvation but was increased after foliar application of BL (Fig. 6B). As compromised formation of autophagosomes promotes the accumulation of ubiquitinated protein aggregates under abiotic stresses (Zhou et al., 2013; Wang et al., 2015), we then determined the changes in insoluble protein content. While BL treatment did not affect the levels of insoluble protein aggregates under N-sufficient conditions (Fig. 6C), N starvation increased the levels of insoluble protein by 103.2% in the absence of BL and by 57.5% in the presence of BL (Fig. 6C). To determine whether these insoluble proteins were ubiquitinated, we isolated total, soluble, and insoluble proteins and separated them by SDS-PAGE to analyze their ubiquitination using an anti-ubiquitin monoclonal antibody. No significant differences in the total, soluble, and insoluble protein levels were observed in any of the plants under N-sufficient conditions (Fig. 6D). N starvation resulted in a reduced increase in the level of ubiquitinated proteins in BL-treated plants (Fig. 6D). BL increased the number of MDC-stained autophagosomes under N-starvation conditions but not under N-sufficient conditions (Fig. 6, E and F). In addition, there was an increased accumulation of Atg8-PE in response to BL under N-starvation conditions (Fig. 6G). Figure 6. Open in new tabDownload slide Role of BRs in the response to N starvation in tomato leaves. A, Exogenous BL increased tolerance to N starvation in tomato plants. Two-week-old plants were transferred to N-free medium for 14 d. Bar = 10 cm. B, The chlorophyll content of the fourth expanded leaves was determined immediately on day 14 under N starvation. FW, Fresh weight. C, Exogenous BL alleviated the accumulation of insoluble proteins under N starvation. Leaf tissues were collected on day 14 under N starvation for the preparation of total, soluble, and insoluble proteins as described in “Materials and Methods.” Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined to calculate the percentages of insoluble proteins to total proteins. D, Exogenous BL inhibited the ubiquitination of insoluble protein aggregates under N starvation. Proteins from the starting homogenates (T), first supernatant fractions (S), and last pellet fractions (P) were subjected to SDS-PAGE and probed with an anti-ubiquitin monoclonal antibody. E, MDC-stained autophagosomes in BL-treated and control plants on day 7 under N starvation. MDC-stained autophagosomes are shown in green. Bars = 20 μm. F, Relative autophagic activity normalized to the activity of the wild-type (WT) control plants in E. The number of MDC-stained autophagosomes per image was quantified to calculate the autophagic activity relative to wild-type control plants, which was set to 1. More than 20 images for each treatment were used for the quantification. G, Atg8 protein levels in the leaves of BL-treated and control plants on day 7 under N starvation. The nonlipidated and lipidated forms of Atg8 are indicated by Atg8 and Atg8-PE, respectively. Actin was used as a loading control for the western-blot analysis. The results in B, C, and F represent means ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. Figure 6. Open in new tabDownload slide Role of BRs in the response to N starvation in tomato leaves. A, Exogenous BL increased tolerance to N starvation in tomato plants. Two-week-old plants were transferred to N-free medium for 14 d. Bar = 10 cm. B, The chlorophyll content of the fourth expanded leaves was determined immediately on day 14 under N starvation. FW, Fresh weight. C, Exogenous BL alleviated the accumulation of insoluble proteins under N starvation. Leaf tissues were collected on day 14 under N starvation for the preparation of total, soluble, and insoluble proteins as described in “Materials and Methods.” Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined to calculate the percentages of insoluble proteins to total proteins. D, Exogenous BL inhibited the ubiquitination of insoluble protein aggregates under N starvation. Proteins from the starting homogenates (T), first supernatant fractions (S), and last pellet fractions (P) were subjected to SDS-PAGE and probed with an anti-ubiquitin monoclonal antibody. E, MDC-stained autophagosomes in BL-treated and control plants on day 7 under N starvation. MDC-stained autophagosomes are shown in green. Bars = 20 μm. F, Relative autophagic activity normalized to the activity of the wild-type (WT) control plants in E. The number of MDC-stained autophagosomes per image was quantified to calculate the autophagic activity relative to wild-type control plants, which was set to 1. More than 20 images for each treatment were used for the quantification. G, Atg8 protein levels in the leaves of BL-treated and control plants on day 7 under N starvation. The nonlipidated and lipidated forms of Atg8 are indicated by Atg8 and Atg8-PE, respectively. Actin was used as a loading control for the western-blot analysis. The results in B, C, and F represent means ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. To examine whether BZR1-modulated autophagy is involved in nutrient remobilization, we used wild-type plants, BZR1OE plants, and BZR1-1D-overexpressing (BZR1-1DOE) plants, which contain a BZR1 mutated in the putative Pro-, Glu-, Ser-, and Thr-rich domain (Tang et al., 2011), to promote the accumulation of dephosphorylated BZR1 (Supplemental Fig. S7) and the BR biosynthetic mutant d^im to detect their tolerance to N starvation. As shown in Figure 7A, the dephosphorylated band of BZR1 was increased gradually in the first 5 d under N starvation but began to decrease on day 7. Except for d^im plants, all other plants exhibited similar growth phenotypes under N-sufficient conditions (Fig. 7B). In contrast, N starvation significantly inhibited plant growth (Fig. 7B). After 7 d of N starvation, the cotyledons of BZR1-silenced plants began to lose their green color, but TRV plants remained green. After 14 d, the fourth fully expanded leaves of TRV-BZR1 plants showed chlorosis, but TRV plants showed less severe symptoms than TRV-BZR1 plants (Fig. 7B). Similarly, the leaves of wild-type plants were light green, while the leaves of BZR1OE and BZR1-1DOE plants remained green until day 14 after N starvation, but the leaves of d^im plants showed chlorosis (Fig. 7B). To confirm the observed phenotype, the chlorophyll contents in TRV, TRV-BZR1, wild-type, BZR1OE, BZR1-1DOE, and d^im plants were measured. The chlorophyll content was not significantly different in these plants, except for BZR1-1DOE plants, which showed higher chlorophyll contents under N-sufficient conditions (Fig. 7C). After 14 d of N-starvation treatment, the chlorophyll content in TRV plants decreased by 61.6% compared with that in TRV control plants, while that in BZR1-silenced plants was 70.7% lower than that in TRV-BZR1 control plants (Fig. 7C). Although N starvation decreased the chlorophyll content in wild-type, BZR1OE, and BZR1-1DOE plants, its content was far higher than that in wild-type plants (Fig. 7C). Figure 7. Open in new tabDownload slide Role of BZR1 in the response to N starvation in tomato leaves. A, N starvation induced the dephosphorylation of BZR1. The phosphorylated and dephosphorylated forms of BZR1 are indicated by pBZR1 and dBZR1, respectively. Two-week-old plants were transferred to N-deficient nutrient solution to collect the leaf samples at the indicated time points. Total proteins were isolated, subjected to 12% SDS-PAGE, and probed with an anti-HA monoclonal antibody. Actin was used as a loading control for the western-blot analysis. B, Tolerance to N starvation in TRV, TRV-BZR1, wild type (WT), BZR1OE, BZR1-1DOE, and d^im plants. The plants were transferred to N-free medium for 14 d. Bars = 10 cm. C, The chlorophyll content of the fourth expanded leaves was determined immediately after 14 d of control or N-deficient treatment in TRV and TRV-BZR1 plants or wild-type, BZR1OE, BZR1-1DOE, and d^im plants. FW, Fresh weight. D, MDC-stained autophagosomes in the leaves of TRV, TRV-BZR1, wild type, BZR1OE, BZR1-1DOE, and d^im plants. The plants were transferred to N-free medium for 7 d, and the leaves were stained with MDC and visualized by confocal microscopy. MDC-stained autophagosomes are shown in green. Bars = 20 μm. E, Relative autophagic activity normalized to the activity of the TRV or wild-type control plants in D. The number of MDC-stained autophagosomes per image was quantified to calculate the autophagic activity relative to TRV or wild-type control plants, which was set to 1. More than 20 images for each treatment were used for the quantification. F and G, Atg8 protein levels in the leaves of TRV, TRV-BZR1, wild type, BZR1OE, BZR1-1DOE, and d^im plants on day 7 under N starvation. The nonlipidated and lipidated forms of Atg8 are indicated by Atg8 and Atg8-PE, respectively. Actin was used as a loading control for the western-blot analysis. The results in C and E represent means ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. 1# represents a line of BZR1OE plants, and 6# represents a line of BZR1-1DOE plants. Figure 7. Open in new tabDownload slide Role of BZR1 in the response to N starvation in tomato leaves. A, N starvation induced the dephosphorylation of BZR1. The phosphorylated and dephosphorylated forms of BZR1 are indicated by pBZR1 and dBZR1, respectively. Two-week-old plants were transferred to N-deficient nutrient solution to collect the leaf samples at the indicated time points. Total proteins were isolated, subjected to 12% SDS-PAGE, and probed with an anti-HA monoclonal antibody. Actin was used as a loading control for the western-blot analysis. B, Tolerance to N starvation in TRV, TRV-BZR1, wild type (WT), BZR1OE, BZR1-1DOE, and d^im plants. The plants were transferred to N-free medium for 14 d. Bars = 10 cm. C, The chlorophyll content of the fourth expanded leaves was determined immediately after 14 d of control or N-deficient treatment in TRV and TRV-BZR1 plants or wild-type, BZR1OE, BZR1-1DOE, and d^im plants. FW, Fresh weight. D, MDC-stained autophagosomes in the leaves of TRV, TRV-BZR1, wild type, BZR1OE, BZR1-1DOE, and d^im plants. The plants were transferred to N-free medium for 7 d, and the leaves were stained with MDC and visualized by confocal microscopy. MDC-stained autophagosomes are shown in green. Bars = 20 μm. E, Relative autophagic activity normalized to the activity of the TRV or wild-type control plants in D. The number of MDC-stained autophagosomes per image was quantified to calculate the autophagic activity relative to TRV or wild-type control plants, which was set to 1. More than 20 images for each treatment were used for the quantification. F and G, Atg8 protein levels in the leaves of TRV, TRV-BZR1, wild type, BZR1OE, BZR1-1DOE, and d^im plants on day 7 under N starvation. The nonlipidated and lipidated forms of Atg8 are indicated by Atg8 and Atg8-PE, respectively. Actin was used as a loading control for the western-blot analysis. The results in C and E represent means ± se. Means with the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Three independent experiments were performed, with similar results. 1# represents a line of BZR1OE plants, and 6# represents a line of BZR1-1DOE plants. To further estimate the role of BZR1 in the formation of autophagosomes under N starvation, we examined the expression of BZR1-regulated ATG2 and ATG6 genes. The expression levels of ATG2 and ATG6 in BZR1-1DOE plants were higher than those in wild-type plants, while both gene expression levels in the plants, except for BZR1-1DOE plants, did not differ from each other under N-sufficient conditions (Supplemental Fig. S8). N starvation significantly increased the transcript levels of ATG2 and ATG6 in TRV and wild-type plants, but this effect was compromised in BZR1-silenced and d^im plants (Supplemental Fig. S8). Importantly, the expression levels of both genes in BZR1OE and BZR1-1DOE plants increased significantly compared with those in wild-type plants at 7 d under N starvation (Supplemental Fig. S8B). Furthermore, we found that the number of MDC-stained autophagosomes in BZR1-1DOE plants was higher than that in wild-type plants, while no significant difference was observed among TRV, TRV-BZR1, wild-type, BZR1OE, and d^im plants under N-sufficient conditions (Fig. 7, D and E). However, the numbers of MDC-stained autophagosomes increased by 21.3- to 24.5-fold in TRV and wild-type plants on day 7 of N starvation (Fig. 7, D and E). The formation of autophagosomes in BZR1-silenced and d^im plants was suppressed significantly after 7 d of N starvation (Fig. 7, D and E). In contrast, the number of MDC-stained autophagosomes increased significantly in BZR1OE and BZR1-1DOE plants compared with wild-type plants (Fig. 7, D and E). While N starvation increased the level of Atg8-PE, BZR1 silencing dramatically suppressed the accumulation of Atg8-PE compared with that in TRV plants (Fig. 7, F and G). Importantly, BZR1OE and BZR1-1DOE plants had higher abundance while d^im plants had lower abundance of Atg8-PE than wild-type plants under N-starvation conditions (Fig. 7G). These results indicate that BR induces the formation of autophagosomes, which then engulf the ubiquitinated protein aggregates for reuse, resulting in increased resistance to N starvation. DISCUSSION Over the past two decades, studies on autophagy in plants have established its crucial role in growth and development and the response to abiotic and biotic stresses (Bassham et al., 2006). However, our understanding of the mechanism and regulation of autophagy in plants remains obscure. In this study, we demonstrated that BR, a vital phytohormone associated with plant growth, development, and stress responses, contributed to the formation of autophagosomes in tomato. BZR1, a downstream transcription factor of the BR signal transduction pathway, acted as an activator of autophagic genes to promote the formation of autophagosomes, while BR-induced autophagy was involved in N remobilization. This study provides evidence for the mechanisms of the BR-mediated regulation of autophagy in plants. BRs, a class of essential plant-specific steroidal phytohormones, play important roles in plant growth, development, and responses to various abiotic and biotic stresses (Yang et al., 2011; Choudhary et al., 2012). BR-deficient and BR-insensitive mutants usually exhibit severe growth defects, including short petioles and hypocotyls, delayed flowering and leaf senescence, and reduced male fertility (Szekeres et al., 1996; Kim et al., 2005). Treatment with BR enhances tolerance to photooxidative and cold stresses in cucumber (Cucumis sativus; Xia et al., 2009). Similarly, exogenous BR treatment also increases tolerance to oxidative and heat stress in tomato, which is associated with the accumulation of apoplastic hydrogen peroxide and the activation of MPK1/2 (Zhou et al., 2014a). Overexpressing the key BR biosynthetic gene AtDWF4 increases tolerance to cold, dehydration, and heat stresses (Divi and Krishna, 2010; Sahni et al., 2016). Moreover, d^im plants have been found to have higher while DWFOE plants have lower levels of oxidized proteins and membrane lipid peroxidation in response to chilling stress (Xia et al., 2018). Notably, abiotic stresses, such as heat and drought, result in severe damage to cellular components, including protein denaturation and aggregation (Vinocur and Altman, 2005), which are recognized by ubiquitin and degraded via autophagy for reuse (Zhou et al., 2013). In recent years, autophagy has been demonstrated to be involved in the responses to various abiotic stresses (Bassham, 2007). Our previous study showed that atg5 and atg7 mutants are more sensitive to heat, oxidative, salt, and drought stresses in Arabidopsis than wild-type plants (Zhou et al., 2013). Furthermore, silencing of ATG10 or ATG18f compromises the tolerance to drought stress in tomato (Wang et al., 2015). In this study, we found that enhanced levels of BR, either through exogenous application or endogenous manipulation, induced the formation of autophagosomes in tomato leaves (Fig. 1; Supplemental Fig. S1). Thus, the BR signaling pathway might induce autophagy for the degradation of denatured and misfolded proteins to increase stress tolerance. Indeed, exogenous application of BL increased the tolerance to N starvation along with increased formation of autophagosomes and inhibited the accumulation of insoluble protein levels (Fig. 6). In insects, 20-hydroxyecdysone, a steroid hormone, plays a critical role in activating autophagy (Yin and Thummel, 2005; Ryoo and Baehrecke, 2010). For example, injection of 20-hydroxyecdysone increases the expression of ATGs and inhibits the activity of TOR complex 1, leading to the induction of autophagy in the fat body of silkworm (Bombyx mori; Tian et al., 2013). BR and 20-hydroxyecdysone are both steroidal hormones with similar chemical structures and induce autophagy. These results imply that the up-regulation of ATGs and the induction of autophagy by steroid hormones are conserved in both plants and animals. BZR1, a transcription factor downstream of the BR signaling pathway, regulates the expression of numerous BR-responsive genes (Sun et al., 2010). Recent studies have demonstrated that sugar activates the TOR signaling-dependent autophagic pathway to regulate the degradation of BZR1 to balance growth and carbon availability in Arabidopsis (Zhang et al., 2016). In addition, drought and carbon starvation induce the brassinosteroid insensitive2 phosphorylation of DSK2, a selective autophagic receptor that promotes DSK2 interaction with Atg8, thereby targeting BRI1-EMS-suppressor1 for breakdown with the attenuation of plant growth (Nolan et al., 2017). These results suggest that autophagy is involved in the regulation of BR signaling through the degradation of BZR1 under drought stress and carbon starvation. However, transcriptome analysis with an Affymetrix ATH1 array revealed that numerous stress-related genes, including those involved in protein metabolism and modification, defense responses, and calcium signaling, are BR responsive (Divi et al., 2016). Furthermore, BR signaling-defective mutants were hypersensitive to salt stress (Cui et al., 2012). Exogenous BR treatment not only enhanced plant tolerance to drought and cold stresses but also promoted seed germination under salt stress (Kagale et al., 2007). In addition, primary root growth was inhibited dramatically and the accumulation of anthocyanin was increased significantly in wild-type plants grown on low-phosphate medium, but the roots of bzr1-d mutants grew well and the leaves remained green (Singh et al., 2014), suggesting that BR signal participates in the response to low phosphate availability. These results indicate the multiple functions of BZR1 in response to different stresses. In this study, we found that the highest level of BZR1 dephosphorylation was observed after BL treatment for 12 h, and the expression of BR biosynthesis genes was inhibited (Supplemental Fig. S3), indicating that BL treatment transiently activated BR signaling. In addition, silencing of the BZR1 gene abolished the BL-induced formation of autophagosomes (Fig. 2, A and C; Supplemental Fig. S4), while autophagic activity was higher after BL application in BZR1OE plants than in wild-type plants (Fig. 2, A and D; Supplemental Fig. S4). Yeast one-hybrid and ChIP-qPCR assays showed that BZR1 bound directly to the promoters of the ATG2 and ATG6 genes (Fig. 4, B and C). Furthermore, silencing of the BZR1 gene compromised the induction of ATG2 and ATG6 genes by BL treatment (Fig. 3A). These results indicate that the ATG2 and ATG6 genes are target genes of the BZR1 transcription factor and might directly regulate the expression of both genes to induce autophagy. Autophagy has been shown to play a critical role in nutrient starvation. Arabidopsis atg mutants exhibited premature senescence and were hypersensitive to N and fixed carbon starvation (Doelling et al., 2002; Yoshimoto et al., 2004; Chung et al., 2010). The atg7 and atg9 plants showed accelerated senescence when grown on N-free medium, characterized by the premature chlorosis of the mature rosette leaves (Doelling et al., 2002). Nutrient starvation induces the formation of autophagosomes to engulf the denatured proteins and transfer them to vacuoles for reuse, leading to enhanced N-utilization efficiency. However, autophagy-defective mutants compromise the degradation of proteins and the generation of amino acids (Barros et al., 2017). Similarly, BR treatment increased N uptake, which was associated with increased activity of nitrate reductase and increased levels of free amino acids and soluble proteins (Dalio et al., 2013). Additionally, the total nodule number and the efficiency of N fixation were reduced in BR-insensitive Medicago truncatula mutants (Cheng et al., 2017). These results indicate that BR plays a critical role in N utilization. Consistent with these previous studies, our study showed that BL treatment increased the tolerance to N starvation, which was associated with the increased formation of autophagosomes to degrade the insoluble proteins (Fig. 6). Furthermore, BZR1 silencing increased sensitivity to N starvation and suppressed the expression of ATGs and the formation of autophagosomes (Fig. 7, B, D, and F). However, BZR1OE plants were more tolerant to N deficiency than wild-type plants, which was associated with the accumulation of increased numbers of autophagosomes (Fig. 7, B, D, and G ). We also found that N starvation promoted the accumulation and dephosphorylation of BZR1 in tomato at the early stage of N-free treatment, while the abundance and dephosphorylation of BZR1 gradually decreased after 7 d (Fig. 7A), which was consistent with the results obtained for Arabidopsis under carbon starvation (Zhang et al., 2016; Nolan et al., 2017). These results showed that, at the early stage of N starvation, the accumulation and dephosphorylation of BZR1 was enhanced in plants to promote N recycling and increased cellular energy by inducing the expression of ATG2 and ATG6 and the formation of autophagosomes. As the stress of N starvation progresses, plant growth is inhibited and autophagy may break down BZR1 to balance growth and the stress response, leading to a decrease in its accumulation, as observed in Arabidopsis in response to carbon starvation and drought (Zhang et al., 2016; Nolan et al., 2017). These results suggest that autophagy and BZR1 can regulate each other and that BZR1 plays dual roles under starvation stresses. In summary, in this study, we used comprehensive genetic and molecular tools to provide insights into the role of BR in the regulation of autophagy. Upon the perception of BR by BRI1, BRs activate the downstream signal transduction cascades, resulting in the dephosphorylation and nuclear localization of BZR1, which can induce the expression of ATGs to trigger autophagosome formation. In addition, BR-induced autophagy mediates the response to N deficiency in tomato plants (Fig. 8). This report systematically illustrates the mechanism of autophagy induction by BRs. Figure 8. Open in new tabDownload slide Proposed model for the induction of autophagy by BRs in tomato plants. Upon the perception of BR by BRI1, BR activates the downstream signal transduction cascades, leading to the dephosphorylation and nuclear localization of BZR1, which induces the expression of ATG2 and ATG6 to trigger the formation of autophagosomes. Furthermore, BR-induced autophagy is involved in the response to N starvation. Figure 8. Open in new tabDownload slide Proposed model for the induction of autophagy by BRs in tomato plants. Upon the perception of BR by BRI1, BR activates the downstream signal transduction cascades, leading to the dephosphorylation and nuclear localization of BZR1, which induces the expression of ATG2 and ATG6 to trigger the formation of autophagosomes. Furthermore, BR-induced autophagy is involved in the response to N starvation. MATERIALS AND METHODS Plant Materials and Experimental Design The tomato (Solanum lycopersicum) ‘Condine Red’ genotype was used in all experiments. Germinated seeds were grown in 250-cm3 plastic pots filled with a mixture of peat and vermiculite (2:1, v/v). The plants were watered daily with Hoagland nutrition solution in the chamber. The growth conditions were maintained at 23°C/21°C day/night temperatures with a photoperiod of 14 h at 600 μmol m−2 s−1 photosynthetic photon flux density. For treatment with BL (Sigma-Aldrich B1439), 6-week-old plants were sprayed with 500 nm BL, and the control plants were sprayed with Milli-Q water containing an equal amount of ethanol used for the preparation of BL solution. Twelve hours after BL treatment, the upper first fully expanded leaves were excised to detect autophagic activity, or they were sampled and frozen quickly in liquid N and stored at −80°C before using them for gene expression, protein analysis, and biochemical analysis. For N-starvation experiments, 2-week-old seedlings were grown in N-free liquid medium containing Murashige and Skoog micronutrient salts (Sigma-Aldrich M0529), 3 mm CaCl2, 1.5 mm MgSO4, 1.25 mm KH2PO4, 5 mm KCl, and 2 mm MES (pH 5.7). The plants were sprayed with 500 nm BL or water every 2 d. The autophagic activity was monitored on day 7, and the chlorophyll content and protein levels were measured on day 14 under N-deficient treatment. Total RNA Isolation and Gene Expression Analysis Total RNA was extracted from tomato leaves by using the RNAsimple Total RNA Kit (Tiangen DP419) according to the manufacturer’s instructions. One microgram of total RNA was used to reverse transcribe to cDNA template using the ReverTra Ace qPCR RT Kit (Toyobo FSQ-301). The RT-qPCR assays were performed using the SYBR Green PCR Master Mix (Takara RR420A) in the LightCycler 480 II Real-Time PCR detection system (Roche). The PCR conditions consisted of denaturation at 95°C for 3 min followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 30 s. The Actin and Ubiquitin3 genes were used as internal controls. Gene-specific primers were designed based on cDNA sequences as described in Supplemental Table S1. Relative gene expression was calculated as described previously (Livak and Schmittgen, 2001). MDC Staining Tomato leaves were stained with MDC as described previously (Wang et al., 2015). Briefly, tomato leaves were excised and then immediately vacuum infiltrated with 100 μm MDC (Sigma-Aldrich 30432) for 30 min, followed by two washes with phosphate-buffered saline (PBS; Solarbio P1020). MDC-incorporated structures were excised by a wavelength of 405 nm and detected at 400 to 580 nm in the LSM 780 confocal microscope (Carl Zeiss). TEM Analysis To visualize the accumulation of autophagosomes by TEM, tomato leaves were cut into small pieces (∼1 mm × 4 mm) and fixed with 2.5% (v/v) glutaraldehyde in 0.1 m PBS buffer (pH 7) for 12 h in the dark. Then, they were washed with PBS buffer three times and again fixed in 1% (v/v) osmium tetroxide at room temperature for 2 h; the samples then were dehydrated in a graded ethanol series (30%–100%, v/v) and embedded in Epon 812. Ultrathin sections (70 nm) were prepared on an ultramicrotome (Leica EM UC7) with a diamond knife and collected on Formvar-coated grids. The sections were detected using an H7650 transmission electron microscope (Hitachi) at an accelerating voltage of 75 kV to observe autophagosomes and autophagic bodies. Protein Extraction and Western Blotting For protein extraction, the harvested tomato leaf samples were ground in liquid N and homogenized in the extraction buffer (20 mm HEPES, pH 7.5, 40 mm KCl, 1 mm EDTA, 1% Triton X-100, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 5 mm DTT, and 25 mm sodium fluoride). The soluble, insoluble, and ubiquitinated proteins were detected as described in our previous study (Zhou et al., 2013). The extracted proteins were heated at 95°C for 15 min; this was followed by separation using 10% SDS-PAGE. For Atg8 detection, the denatured proteins were separated on a 13.5% SDS-PAGE gel in the presence of 6 m urea. For western blotting, the proteins on the SDS-PAGE gel were transferred to a nitrocellulose membrane. Then, the membrane was blocked for 1 h in TBS buffer (20 mm Tris, pH 7.5, 150 mm NaCl, and 0.1% Tween 20) with 5% skim milk powder at room temperature and then incubated for 1 h in TBS buffer with 1% BSA (Amresco 0332) containing a mouse anti-HA monoclonal antibody (Pierce 26183), mouse anti-ubiquitin monoclonal antibody (Sigma-Aldrich U0508), rabbit anti-actin polyclonal antibody (Abcam ab197345), or rabbit anti-Atg8 polyclonal antibody (Abcam ab77003 or Agrisera AS142769). After incubation with a goat anti-mouse horseradish peroxidase-linked antibody (Millipore AP124P) or goat anti-rabbit horseradish peroxidase-linked antibody (Cell Signaling Technology 7074), the complexes on the blot were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific 34080) by following the manufacturer’s instructions. Vector Construction and Transformation To obtain the tomato BZR1OE construct, the 981-bp full-length coding DNA sequence (CDS) was amplified with specific primers (Supplemental Table S2) using tomato cDNA as the template. The PCR product was digested with AscI and KpnI and inserted behind the Cauliflower mosaic virus 35S promoter in the plant transformation vector pFGC1008-HA. To obtain the BZR1-1DOE construct, the CDS was amplified using specific primers (Supplemental Table S2) and ligated to pFGC1008-HA vector using the ClonExpress MultiS One Step Cloning Kit (Vazyme C113-01). The resulting plasmids were transformed into Agrobacterium tumefaciens strain EHA105 and transformed into tomato seeds as described previously (Fillatti et al., 1987). Transgenic plants overexpressing the BZR1 and BZR1-1D transgene were identified by RT-qPCR (Supplemental Fig. S2B). Two independent homozygous lines of the T2 progeny were used in the study. Virus-Induced Gene Silencing Constructs and A. tumefaciens-Mediated Virus Infection The virus-induced gene silencing (VIGS) constructs for silencing of the BZR1, ATG2, and ATG6 genes were generated by PCR amplification using specific primers (Supplemental Table S3), digested with SacI and XhoI, and ligated into the same sites in TRV2. The resulting plasmid was transformed into A. tumefaciens strain GV3101. A. tumefaciens-mediated virus infection was performed as described previously (Ekengren et al., 2003). The plants were kept at 22°C and used for experiments after A. tumefaciens infiltration for 3 weeks. Leaflets in the middle of the fifth fully expanded leaves, which showed about 20% to 40% transcript levels of control plants, were used. Yeast One-Hybrid Assay The yeast one-hybrid experiment was performed as described previously (Ravindran et al., 2017). The promoter sequences of ATGs and the CDS of BZR1 were amplified using specific primers (Supplemental Table S4) and ligated into the pAbAi and pGADT7 vectors, respectively. To generate the mutant of the E-boxes, the sequence CANNTG was replaced by TCNNAA using the Fast MultiSite Mutagenesis System (TransGen FM201-01) according to the manufacturer’s instructions, and the primers used for plasmid construction are listed in Supplemental Table S5. All constructs were checked by DNA sequencing. The linearized constructs containing ATG promoter fragments in pAbAi were integrated into the genome of the Y1H Gold yeast strain, and either BZR1-AD or an empty AD vector was transformed into each. The transformed yeast cells were selected on Leu− plates supplemented with 100 ng mL−1 aureobasidin A to detect DNA-protein interactions. ChIP ChIP experiments were performed using the EpiQuik Plant ChIP Kit (Epigentek P-2014) according to the manufacturer’s instructions. Briefly, approximately 1 g of leaf tissue was harvested from BL-treated 35S-BZR1-HA and wild-type plants. Chromatin was immunoprecipitated with an HA antibody (Pierce 26183), and goat anti-mouse IgG (Millipore AP124P) was used as the negative control. ChIP-qPCR was performed with primers specific for the ATG2, ATG5, and ATG6 promoters (Supplemental Table S6). Chlorophyll Content The chlorophyll in tomato leaves was extracted in 80% (v/v) acetone, and its content was analyzed spectrophotometrically as described previously (Chung et al., 2010). Statistical Analysis At least five independent replicates were used for each determination. Statistical analysis of the bioassays was performed using the SPSS for Windows version 18.0 (CoHort Software) statistical package. Experimental data were analyzed with Duncan’s multiple range test at P < 0.05. Accession Numbers Sequence data from this article can be found in Solgenomics data libraries (http://solgenomics.net/) according to the accession numbers listed in Supplemental Tables S1 and S2. Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Relevance of endogenous BRs inducing autophagy in tomato leaves. Supplemental Figure S2. Relative mRNA abundance of BZR1 in VIGS, BZR1OE, and BZR1-1DOE plants. Supplemental Figure S3. BL induced the dephosphorylation of BZR1 and inhibited the expression of BR biosynthetic genes in BZR1OE plants. Supplemental Figure S4. Visualization of the accumulation of autophagosomes in BZR1-silenced and BZR1OE plants with BL treatment by TEM. Supplemental Figure S5. BZR1 binds to the promoters of ATGs in vitro. Supplemental Figure S6. Relative mRNA abundance of ATG2 and ATG6 in ATG2- and ATG6-silenced wild-type or BZR1OE plants. Supplemental Figure S7.. Induction of the dephosphorylation of BZR1 by BL in BZR1OE and BZR1-1DOE plants. Supplemental Figure S8. Expression of ATG2 and ATG6 in BZR1-silenced, BZR1OE, BZR1-1DOE, and d^im plants. Supplemental Table S1. Primers used for RT-qPCR assays. Supplemental Table S2. Primers used for the construction of BZR1OE and BZR1-1DOE vectors. Supplemental Table S3. Primers used for VIGS vector construction. Supplemental Table S4. Primers used for yeast one-hybrid assays. Supplemental Table S5. Primers used for the construction of ATG2 and ATG6 promoter mutant vectors. Supplemental Table S6. Primers used for ChIP-qPCR assays. LITERATURE CITED Albrecht C , Boutrot F, Segonzac C, Schwessinger B, Gimenez-Ibanez S, Chinchilla D, Rathjen JP, de Vries SC, Zipfel C ( 2012 ) Brassinosteroids inhibit pathogen-associated molecular pattern-triggered immune signaling independent of the receptor kinase BAK1 . 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Front Plant Sci 5 : 174 Google Scholar PubMed OpenURL Placeholder Text WorldCat Zientara-Rytter K , Lukomska J, Moniuszko G, Gwozdecki R, Surowiecki P, Lewandowska M, Liszewska F, Wawrzyńska A, Sirko A ( 2011 ) Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors . Autophagy 7 : 1145 – 1158 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the National Key Research and Development Program of China (2018YFD1000800) and the National Natural Science Foundation of China (31872089, 31430076, and 31801902). 2 These authors contributed equally to the article. 3 Author for contact: jie@zju.edu.cn. 4 Senior author. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jie Zhou (jie@zju.edu.cn). Y.W. and J.Z. planned and designed the research; Y.W. and J.-J.C. performed experiments and analyzed data; K.-X.W., X.-J.X., K.S., Y.-H.Z., and J.-Q.Y. performed molecular cloning and analyzed data; Y.W. and J.Z. wrote the article; all authors reviewed, revised, and approved the article. www.plantphysiol.org/cgi/doi/10.1104/pp.18.01028 © 2019 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - BZR1 Mediates Brassinosteroid-Induced Autophagy and Nitrogen Starvation in Tomato
JF - PLANT PHYSIOLOGY
DO - 10.1104/pp.18.01028
DA - 2019-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/bzr1-mediates-brassinosteroid-induced-autophagy-and-nitrogen-R6QIkQ60x7
SP - 671
EP - 685
VL - 179
IS - 2
DP - DeepDyve
ER -